Complement factors C1q, C3 and C5b-9 in the posterior sclera of guinea pigs with negative lens-defocused myopia

· AIM: To investigate the expression of complement factors in the posterior scleral fibroblasts of guinea pigs with negative lens-defocused myopia.· METHODS: Eighteen guinea pigs were assigned randomly to two groups: the negative lens-defocused group(NLD group, n =9) and the normal control without treatment group(NC group, n =9). The effect of myopic induction was compared in three subgroups: eyes treated with a-10.00 D negative lens in the NLD group(NL group), eyes treated with a plano(0 D) lens in the NLD group(PL group), and untreated right eyes in the NC group(NC group). The following analyses were conducted at four weeks: examination of the refractive error via retinoscopy, assessment of complement C5b-9expression in the posterior scleral fibroblasts using immunohistochemistry, and measurements of complement C1 q and C3 protein levels in the posterior sclera by Western blot.·RESULTS: After an induction period of four weeks, a significant myopic shift was detected in the eyes of the NL group, relative to that of the PL and NC groups(P 【0.05). Data analysis showed a significant increase in the percentage of C5b-9 immunopositive fibroblasts in the posterior sclera of the NL group eyes, compared to the PL group(q =11.50, P 【0.001). Significantly higher levels of C1q(q =4.94, P =0.01) and C3(q =4.07, P =0.03)protein were detected in the posterior sclera of NL group eyes, compared to the PL group. There were no significant difference between the PL and NC groups for C5b-9(q =2.44, P =0.10), C1q(q =1.55, P =0.53) and C3(q =0.98, P =0.77) in the posterior sclera.·CONCLUSION: The data from present study provide evidence of the up-regulation of C5b-9, C1 q and C3 in the posterior scleral fibroblasts in a NLD myopic animal model. The results suggest that the complement system may be involved in the development of myopia.

Interleukin-1 receptor associated kinase 2 is a functional downstream regulator of complement factor D that controls mitochondrial fitness in diabetic cardiomyopathy

Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.

Complement factor Ⅰ knockdown inhibits colon cancer development by affecting Wnt/β-catenin/c-Myc signaling pathway and glycolysis

BACKGROUND Colon cancer(CC)occurrence and progression are considerably influenced by the tumor microenvironment.However,the exact underlying regulatory mechanisms remain unclear.AIM To investigate immune infiltration-related differentially expressed genes(DEGs)in CC and specifically explored the role and potential molecular mechanisms of complement factor I(CFI).METHODS Immune infiltration-associated DEGs were screened for CC using bioinformatics.Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines.Stable CFI-knockdown HT29 and HCT116 cell lines were constructed,and the diverse roles of CFI in vitro were assessed using CCK-8,5-ethynyl-2’-deoxyuridine,wound healing,and transwell assays.Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice.Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting.RESULTS Six key immune infiltration-related DEGs were screened,among which the expression of CFI,complement factor B,lymphoid enhancer binding factor 1,and SRY-related high-mobility-group box 4 was upregulated,whereas that of fatty acid-binding protein 1,and bone morphogenic protein-2 was downregulated.Furthermore,CFI could be used as a diagnostic biomarker for CC.Functionally,CFI silencing inhibited CC cell proliferation,migration,invasion,and tumor growth.Mechanistically,CFI knockdown downregulated the expression of key glycolysis-related proteins(glucose transporter type 1,hexokinase 2,lactate dehydrogenase A,and pyruvate kinase M2)and the Wnt pathway-related proteins(β-catenin and c-Myc).Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway.CONCLUSION The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway,indicating that it could serve as a promising target for therapeutic intervention in CC.

Association of C-reactive protein and complement factor H gene polymorphisms with risk of lupus nephritis in Chinese population

BACKGROUND Complement overactivation is a major driver of lupus nephritis(LN).Impaired interactions of C-reactive protein(CRP)with complement factor H(CFH)have been shown as a pathogenic mechanism that contributes to the overactivation of complement in LN.However,genetic variations of neither CRP nor CFH show consistent influences on the risk of LN.AIM To examine whether genetic variations of CRP and CFH in combination can improve the risk stratification in Chinese population.METHODS We genotyped six CRP single nucleotide polymorphisms(SNPs)(rs1205,rs3093062,rs2794521,rs1800947,rs3093077,and rs1130864)and three CFH SNPs(rs482934,rs1061170,and rs1061147)in 270 LN patients and 303 healthy subjects.RESULTS No linkage was found among CRP and CFH SNPs,indicating lack of genetic interactions between the two genes.Moreover,CRP and CFH SNPs,neither individually nor in combination,are associated with the risk or clinical manifestations of LN.Given the unambiguous pathogenic roles of the two genes.CONCLUSION These findings suggest that the biological effects of most genetic variations of CRP and CFH on their expressions or activities are not sufficient to influence the disease course of LN.

Association between complement factor I gene polymorphisms and the risk of age-related macular degeneration:a Meta-analysis of literature

AIM： To systematically review the association between complement factors I （CFI） polymorphisms and age- related macular degeneration （AMD） and to explore whether CFI polymorphisms are associated with AMD, METHODS： Meta-analysis of articles published from 1995 to January 2015 of articles involved with AMD and polymorphisms of the CFI gene. Eligible data were pooled in a Meta-analysis, analyzing using STATA software （version 12.0）, Review Manager （version 5.2） and different models based on the heterogeneity of effect sizes. Egger＇s test, Begg＇s rank correlation methods were used to evaluate for publication bias.~ RESULTS： Thirteen articles were eligible, describing two loci polymorphisms of the CFI gene （of which 12 articles focus on rs10033900T〉C and 3 articles focus on rs2285714C〉T）. For rs10033900T〉C, the results of our study revealed that having a mutant allele C, TC, CC and TC＋CC was associated with a decreased risk of AMD in all population groups studied （C versus T models, OR=0.84, 95%Ch 0.72-0.99, P=0.04; TC versus TT models OR= 0.89, 95%Ch 0.88-0.99, P=0.04;CC versus ＂1-1＂ models, OR=0.76, 95%Ch 0.60-0.98, P=-0.03; TC＋CC versus TT models, OR=0.81, 95%Ch0.65-0.99, P=0.04）. We found that C allele were related to lower AMD risk in the Caucasian population by subgroup analysis, but there was no association with AMD under the allele and genotypes comparison in Asian studies. For rs2285714 C〉T, the TC, TT genotypes contributed to a higher risk of AMD, compared with the CC carriers and CT＋CC （OR=1.34, 95%Ch 1.09-1.63, P=0.004; OR=1.50, 95%Ch 1.25-1.80, P〈0.0001）. CONCLUSION： This Meta-analysis suggests that CFI rs10033900T〉C and rs2285714C〉T polymorphisms may contribute to AMD.

Association between complementary factor H Y402H polymorphisms and age-related macular degeneration in Chinese: Systematic review and meta-analysis

AIM: Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world and complement factor H (CFH) polymorphism has been found to associate with the AMD. To investigate whether the Y402H variant in CFH is associated with AMD in Chinese populations, a systematic review and meta-analysis were performed to estimate the magnitude of the gene effect and the possible mode of action. METHODS: A meta-analysis was performed using data available from ten case-control studies assessing association between the CFH Y402H polymorphism and AMD in Chinese populations involving 1538 AMD. Data extraction and study quality assessment were performed in duplicate. Summary odds ratios (ORs) and 95% confidence intervals (CIs) an allele contrast and genotype contrast were estimated usingfixed- effects models. The Q-statistic test was used to assess heterogeneity, and Funnel plot was used to evaluate publication bias. RESULTS: Seven of ten case-control studies were neovascular AMD, and few studies came from west and north of China. There was strong evidence for association between CFH and AMD in Chinese population, with those having risk allele C 2.35 times more likely to have AMD than subjects with T allele. Evidence of publication bias was not observed in our meta-analysis. CONCLUSION: This meta-analysis summarizes the strong evidence for an association between CFH and AMD in Chinese and indicates each C allele increasing the odds of AMD by 2.33-fold. But more evidences about the relation between CFH polymorphism and different type of Chinese AMD from various district were needed.

Complement factor B polymorphism(rs641153) and susceptibility to age-related macular degeneration: evidence from published studies

AIM:To determine whether single nucleotide polymorphism(SNP)rs641153 is associated with the risk of age-related macular degeneration(AMD),we performed a systematic meta-analysis of 15 eligible studies.SNP in the complement factor B(CFB)gene is considered to have significant association with AMD susceptibility,but there is great discrepancy in these results.METHODS:The eligible studies were identified by searching the databases of PubMed,EMBASE,and Web of Science.Odds ratios(ORs)with 95%confidence intervals(CIs)were used to assess the association.All data were analyzed using Stata software.RESULTS:The association between rs641153 and AMD risk was statistically significant under the homozygous model(AA vs GG:OR=0.26,95%CI=0.15-0.45,P_h=0.973,/~2=0.0%,fixed effects),dominant model(AA+GA vsGG:OR=0.49,95%CI=0.40-0.59,P_h=0.004,/~2=56.4%,random effects)and recessive model(AA vs GA+GG:OR=0.30,95%CI=0.17-0.51,R_n=0.983,I^2=0.0%,fixed effects).The same results were also observed in the stratified analyses by ethnicity,source of control and sample size.CONCLUSION:Our meta-analysis suggests that rs641153 in the CFB gene may play a protective role in AMD susceptibility,the late AMD in particular,both in Caucasians and in Asians.

Novel Mechanistic Interplay between Products of Oxidative Stress and Components of the Complement System in AMD Pathogenesis

Age-related macular degeneration (AMD) is a leading cause of vision loss affecting tens of millions of elderly worldwide. Early AMD includes soft drusen and pigmentary changes in the retinal pigment epithelium (RPE). As people age, such soft confluent drusen can progress into two forms of advanced AMD, geographic atrophy (GA, or dry AMD) or choroidal neovascularization (CNV, or wet AMD) and result in the loss of central vision. The exact mechanism for developing early AMD and progressing to advanced stage of disease is still largely unknown. However, significant evidence exists demonstrating a complex interplay of genetic and environmental factors as the cause of AMD progression. Together, complement factor H (CFH) and HTRA1/ARMS polymorphisms contribute to more than 50% of the genetic risk for AMD. Environmentally, oxidative stress from activities such as smoking has also demonstrated a powerful contribution to AMD progression. To extend our previous finding that genetic polymorphisms in CFH results in OxPLs and the risk-form of CFH (CFH Y402H) has reduced affinity for oxidized phospholipids, and subsequent diminished capacity which subsequently diminishes the capability to attenuate the inflammatory effects of these molecules, we compared the binding properties of CFH and CFH related protein 1 (CFHR1), which is also associated with disease risk, to OxPLs and their effects on modulating inflammation and lipids uptake. As both CFH-402H and CFHR1 are associated with increased risk to AMD, we hypothesized that like CFH-402H, CFHR1 contribution to AMD risk may also be due to its diminished affinity for OxPLs. Interestingly, we found that association of CFHR1 with OxPLs was not statistically different than CFH. However, binding of CFHR1 did not elicit the same protective benefits as CFH in that both inflammation and lipid uptake are unaffected by CFHR1 association with OxPLs. These findings demonstrate a novel and interesting complexity to the potential interplay between the complement system and oxidative stress byproducts, such as OxPLs, in the mechanistic contribution to AMD. Future work will aim to identify the molecular distinctions between CFH and CFHR1 which confer protection by the former, but not latter molecules. Understanding the molecular domains necessary for protection could provide interventional insights in the generation of novel therapeutics for AMD and other diseases associated with oxidative stress.

Atypical hemolytic-uremic syndrome due to complement factor Ⅰ mutation

Atypical hemolytic-uremic syndrome(a HUS) is a rare disease of complement dysregulation leading to thrombotic microangiopathy(TMA). Renal involvement and progression to end-stage renal disease are common in untreated patients. We report a 52-year-old female patient who presented with severe acute kidney injury, microangiopathic hemolytic anemia, and thrombocytopenia. She was managed with steroid, plasma exchange, and dialysis. Kidney biopsy shows TMA and renal cortical necrosis. Genetic analysis reveals heterozygous complement factor Ⅰ(CFI) mutation. Eculizumab was initiated after 3 mo of presentation, continued for 9 mo, and stopped because of sustained hematologic remission, steady renal function, and cost issues. Despite this, the patient continued to be in hematologic remission and showed signs of renal recovery, and peritoneal dialysis was stopped 32 mo after initiation. We report a case of a HUS due to CFI mutation, which, to the best of our knowledge, has not been reported before in Saudi Arabia. Our case illustrates the challenges related to the diagnosis and management of this condition, in which a high index of suspicion and prompt treatment are usually necessary.

Complement factor H in molecular regulation of angiogenesis

Angiogenesis,the process of formation of new capillaries from existing blood vessels,is required for multiple physiological and pathological processes.Complement factorH(CFH)is a plasma protein that inhibits the alternative pathway of the complement system.Loss of CFH enhances the alternative pathway and increases complement activation fragments with pro-angiogenic capacity,including complement 3a,complement 5a,and membrane attack complex.CFH protein contains binding sites for C-reactive protein,malondialdehyde,and endothelial heparan sulfates.Dysfunction of CFH prevents its interaction with these molecules and initiates pro-angiogenic events.Mutations in the CFH gene have been found in patients with age-related macular degeneration characterized by choroidal neovascularization.The Cfh-deficient mice show an increase in angiogenesis,which is decreased by administration of recombinant CFH protein.In this review,we summarize the molecular mechanisms of the anti-angiogenic effects of CFH and the regulatory mechanisms of CFH expression.The therapeutic potential of recombinant CFH protein in angiogenesisrelated diseases has also been discussed.

Update on hemolytic uremic syndrome:Diagnostic and therapeutic recommendations

Hemolytic uremic syndrome （HUS） is a rare disease. In this work the authors review the recent findings on HUS, considering the different etiologic and patho-genetic classifications. New findings in genetics and, in particular, mutations of genes that encode the complement-regulatory proteins have improved our understanding of atypical HUS. Similarly, the comple-ment proteins are clearly involved in all types of thrombotic microangiopathy： typical HUS, atypical HUS and thrombotic thrombocytopenic purpura （TTP）. Fur-thermore, several secondary HUS appear to be related to abnormalities in complement genes in predisposed patients. The authors highlight the therapeutic as-pects of this rare disease, examining both “traditional therapy” （including plasma therapy, kidney and kidney-liver transplantation） and “new therapies”. The latter include anti-Shiga-toxin antibodies and anti-C5 mono-clonal antibody “eculizumab”. Eculizumab has been recently launched for the treatment of the atypical HUS, but it appears to be effective in the treatment of typical HUS and in TTP. Future therapies are in phases Ⅰ and Ⅱ. They include anti-C5 antibodies, which are more purifed, less immunogenic and absorbed orally and, anti-C3 antibodies, which are more powerful, but potentially less safe. Additionally, infusions of recombinant complement-regulatory proteins are a potential future therapy.

Influence of CFH,HTRA1 and ARMS2 polymorphisms in the response to intravitreal ranibizumab treatment for wet age-related macular degeneration in a Spanish population

AIM：To determine whether gene polymorphisms of the major genetic risk loci for age-related macular degeneration（AMD）：ARMS2（rs10490923）,the complement factor H（CFH）（rs1410996） and HTRA1（rs11200638） influence the response to a treatment regimen with ranibizumab for exudative AMD.METHODS：This study included 100 patients（100 eyes）with exudative AMD.Patients underwent a treatment with ranibizumab injections monthly during three months.Reinjections were made when the best corrected visual acuity（BCVA） decrease five letters（ETDRS） or central subfield retinal thickness gained 100 pm in optical coherence tomography image.Genotypes（rs10490923,rs1410996 and rs11200638） were analyzed using TaqMan probes or polymerase chain reaction-restricted fragment length polymorphisms analysis.RESULTS：There were no statistically significant differences in allelic distribution of CFH（rs1410996）,ARMS2（rs10490923） and HTRA1（rs11200638）polymorphisms regarding to response to ranibizumab treatment.CONCLUSION：Ranibizumab treatment response is not related to CFH（rs1410996）,ARMS2（rs10490923） and HTRA1（rs11200638） poymorphisms.

Association of CFH and MAP1LC3B gene polymorphisms with age-related macular degeneration in a high-altitude population

AIM: To evaluate the association of complement factor H(CFH) and microtubule-associated protein 1 light chain 3 beta(MAP1LC3B) gene polymorphisms with the risk of age-related macular degeneration(AMD) in a high-altitude population. METHODS: The study group consisted of 172 participants with symptoms of AMD who were examined and diagnosed between January 2019 and June 2020. The control group was composed of 120 healthy individuals. Each participant was required to provide two milliliters of peripheral blood for DNA extraction. Two single nucleotide polymorphisms(SNPs) of CFH(rs1061170 and rs800292) and two SNPs of MAP1LC3B(rs8044820 and rs9903) were genotyped. The genotypes and allele frequencies of the SNPs in the study and control groups were further compared using Chi-square and Fisher’s exact tests. RESULTS: In a high-altitude population, the nominally significant differences of rs800292 and rs9903’s genotype AG frequencies were observed in the AMD group(P=0.034 and 0.004, respectively). The frequencies of allele G of rs800292 and allele A of rs9903 were also significantly dif ferent in the AMD group compared to the control [(P=0.034, OR=0.70, 95%CI: 0.50-0.98) and(P=0.004, OR=1.60, 95%CI: 1.15-2.22), respectively]. No significant differences in the genotype distributions(P=0.16 and 0.40, respectively) and allele frequencies(P>0.05) of rs1061170 and rs8044820 were observed in the AMD group.CONCLUSION: Genotype AG of rs800292 may be a protective factor for AMD. Conversely, rs9903 seems to be a risk factor for AMD. Therefore, allele G of rs800292 may be a protective factor, and allele A of rs9903, a risk factor for AMD in Qinghai high-altitude population.

Age-related macular degeneration treatment in the era of molecular medicine

Age-related macular degeneration(AMD) is the leading cause of irreversible blindness in the developed world. The quality of life of both patients and families is impacted by this prevalent disease. Previously, macular degeneration had no known effective treatment. Today, vitamins for non-exudative AMD and intravitreal injection of medications for its exudative form are primary forms of current treatment. Modern advances in molecular science give rise to new possibilities of disease management. In the year 2003 the sequencing of the entire human genome was completed. Since that time, genes such as complement factor H, high-temperature requirement factor A1, and age-relateed maculopathy susceptibility 2 have been discovered and associated with a higher risk of AMD. A patient's genetic make-up may dictate the effectiveness of current or future therapeutic options. In addition, utilizing genetic data and incorporating it into new treatments(such as viral vectors) may lead to longer-lasting(or permanent) VEGF blockade and specific targeting of complement related genes. There have also been considerable advances in stem cell directed treatment of AMD. Retinal pigment epithelial(RPE) cells can be derived from human embryonic stem cells, induced pluripotent stem cells, or adult human RPE stem cells. Utilizing animal models of RPE and retinal degeneration, stem cell-derived RPE cells have been successfully implanted into the subretinal space. They have been injected as a cell mass or as a pre-prepared monolayer on a thin membrane. Visual recovery has been demonstrated in a retinal dystrophic rat model. Preliminary data on 2 human subjects also demonstrates possible early visual benefit from transplantation of stem cell-derived RPE. As more data is published, and as differentiation and implantation techniques are optimized, the stabilization and possible improvement of vision in individuals with non-exudative macular becomes a real possibility. We conclude that the technologic advances that continue to unfold in both genetic and stem cell research offer optimism in the future treatment of AMD.

AB009.The age-related macular degeneration genetic-risk promotes pathogenic subretinal inflammation

Mononuclear phagocytes(MP)comprise a family of cells that include microglial cells(MC),monocytes,and macrophages.The subretinal space,located between the RPE and the photoreceptor outer segments,is physiologically devoid of MPs and a zone of immune privilege mediated,among others,by immunosuppressive RPE signals.Age-related macular degeneration(AMD)is a highly heritable major cause of blindness,characterized by a breakdown of the subretinal immunosuppressive environment and an accumulation of pathogenic inflammatory MPs.Studies in mice and humans suggest that the AMD-associated APOE2 isoform promotes the breakdown of subretinal immunosuppression and increased MP survival.Of all genetic factors,variants of complement factor H(CFH)are associated with greatest linkage to AMD.Using loss of function genetics and orthologous models of AMD,we provide mechanistic evidence that CFH inhibits the elimination of subretinal MPs.Importantly,the AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells,indicating a causal link to disease etiology.Pharmacological acceleration of resolution of subretinal inflammation might be a powerful tool for controlling inflammation and neurodegeneration in late AMD.

Genetic variants in three genes and smoking show strong associations with susceptibility to exudative age-related macular degeneration in a Chinese population

Background The present study was undertaken to replicate the associations of representative polymorphisms in three genes （complement factor H （CFH）, complement factor B （BF） and HtrA serine peptidase 1 （HTRA1）） with exudative age-related macular degeneration （AMD） in a Han Chinese population, and to test if the modifiable environmental factors affect AMD susceptibility associated with different type of genotype in these genes. Methods An age, gender and ethnicity matched case-control study was conducted to genotype the representative single neucleotide polymorphisms （SNPs） loci including rs1061170 and rs1410996 in CFH, rs641153 and rs4151667 in BF and rs11200638 in HTRA1 gene in 144 exudative AMD patients and 126 normal controls using PCR-RFLP and direct resequencing. The demographic characteristics and behavioral risk factors were also recorded. Allelic and genotypic associations for individual SNP and joint associations with two loci were performed. The gene-gene and gene-environment interactions were analyzed using multivariate non-conditional Logistic regression analysis. Results The C risk allele frequencies for CFH Y402H （rs1061170） in cases and controls were 12.5% and 5.4% respectively, which were much lower than those in Caucasians （P 〈0.001）. Compared with TT homozygous genotype, the CT heterozygous genotype was positively associated with AMD with odds ratio （OR） of 3.23 （1.36-5.07）. However, the population attributable risk （PAR） of C allele was only 3.3% （1.4%-4.3%）. rs1410996 was also associated with AMD independent of Y402H. The ORs of exudative AMD for individuals carrying one copy risk allele and two copy risk alleles were 2.57 （1.21-5.45） and 4.76 （2.15-10.55） respectively, with correspondent PARs of 28.3% （2.0%-40.5%） and 38.2% （21.8%-45.4%）. rs11200636 in HTRA1 was another susceptible locus for AMD and the risk homozygotes were significantly susceptible for exudutive AMD （OR=3.98, 1.88-8.43） with PAR of 38.9% （24.3%-45.8%）. Education status and cigarette smoking were also related to exudative AMD. After controlling for environmental risk factors, CFH and HTRA1 SNPs were independently associated with exudative AMD, with OR of 3.50 （1.45-8.45） for CT genotype in Y402H, 3.34 （1.33-8.36） for GG genotype in rs1410996 and 3.85 （1.58-9.42） for AA genotype in rs11200638 respectively. The interaction analysis between gene and environmental factors showed that smoking synergistically increased susceptibility of AMD for heterozygotes of rs1410996, with ORinteraction of 7.33 （Pinteraction=0.029）. Conclusions In a Han Chinese population, CFH and HTRA1 polymorphisms appear to be independently and possibly additively hereditary contributors to exudative AMD. Y402H polymorphism conferred a significant but relatively lower contribution in Chinese than in Caucasians with a low frequency of risk allele. The gene-environment interaction may be a best way to encourage those with a high genetic risk to prevent AMD by avoiding modifiable factors until there is effective treatment for AMD.